Data are representative (B, C) or show the summary (A) of five (A, B), and one (C) experiments. column) surface expression. Figure displays staining with specific antibody (solid black line) or control antibody (dashed line). In addition, 5×105 transfectants were incubated with pooled IgG (beriglobin P) followed by staining with an anti-human Ig-specific antiserum (right column, black solid line). Anti-human Ig-specific antiserum alone served as unfavorable control (right column, dashed line). Numbers indicate percent positive cells. (B) Immunoblot analysis of VNP preparations obtained from HEK-293 cells transfected with MoMLV as particle inducing devices and IL-2v constructs as indicated. Anti-p30Gag was used as VNP loading control. Data are representative of four (A) and two (B) impartial experiments.(TIF) pone.0126034.s002.tif (4.9M) GUID:?B4875822-EDFC-4AE7-A346-C5CC205EFFE7 S3 Fig: Strong effector functions induced by IL-2::2Ig(F)GPI asVNP in the absence of bystander activation and proliferation. P14 splenocytes were labeled with CFSE proliferation dye and stimulated with 8 AZD3839 free base g IL-2v asVNP as indicated. (A and B) After 96 hours cells were incubated with PMA/ionomycin in the presence of GolgiStop for 6 hours. Cells were subsequently stained with CD8- and TCR V2-specific mAb followed by intracellular IFN- staining and subjected to flow cytometric analysis. (A) Diagram depicts the fraction of IFN–producing CD8+ TCR V2+-cells obtained from splenocyte cultures. (B) Density plots display FSCN1 intracellular IFN–expression of CD8- TCR V2- lymphocytes relative to cellular proliferation as detected by CFSE-dilution. Markers were set according to unfavorable control staining and non-proliferating cells. (C) Histogram overlays display surface expression of CD107a (LAMP1) (black solid line) or control mAb (shaded grey histogram) of CD8+ TCR V2+ cells after 72 hours of IL-2v asVNP co-culture. Untreated cells and cells stimulated with optimal amounts of LCMV-GP33-41 peptide (100 ng/ml) served as controls. Data are representative (B, C) or show the summary (A) of five (A, B), and one (C) experiments. * p < 0.05. ANOVA and Tukeys multiple comparison test (A).(TIF) pone.0126034.s003.tif (1.3M) GUID:?9EDA7844-DC20-4CCF-B6DF-BBF435C95D15 S4 Fig: Antigen-specific down-regulation of CD127 expression upon co-incubation of CD8+ AZD3839 free base T cells with IL-2v asVNP. (A) Re-expression kinetics of CD127 on IL-2v asVNP stimulated purified P14 CD8+ TCR V2+ T cells. (A) Flow cytometry analysis of CD127 expression (black solid line) on purified P14 CD8+ T cells were stimulated with IL-2::GPI or IL-2::2Ig(F)GPI asVNP and analyzed at indicated time points for surface expression of CD127. Overlay histograms show staining with CD127-specific (black solid line) and Grey shaded histograms show staining with control antibodies. Numbers indicate mean fluorescence intensity. (B) Purified P14 CD8+ T cells were co-incubated with IL-2(A)::GPI and IL-2(A)::2Ig(F)GPI AZD3839 free base asVNP or IL-2::GPI and IL-2::2Ig(F)GPI ansVNP for three days and analyzed for surface expression of the high-affinity IL-7R, CD127. Overlay histograms show staining with CD127-specific (black solid line) and control antibody (grey shaded histogram). Numbers indicate mean fluorescence intensity. (C) Flow cytometry analysis showing expression of the indicated markers on na?ve (shaded grey histograms) and pre-activated (sound black histograms) CD8+CD45.2+ donor cells isolated from the spleens of recipient mice. Data are representative (A-C) of three (except two for ansVNP in B) experiments or of 18 mice (eight per group, except for na?ve (two), IL-2::GPI (three), CD3/CD28 plus IL-2 (five)) analyzed in two independent experiments.(TIF) pone.0126034.s004.tif (3.5M) GUID:?FF7B0349-0EA4-4A12-B91B-0CDC8B0AD40F S1 Table: List of primers. The underlined regions indicate the restriction enzyme (RE) sites(DOCX) pone.0126034.s005.docx (14K) GUID:?D9C3D6F9-13D7-4B3C-90ED-8AB1CEC78F52 S2 Table: List of mAbs. Abbreviations: APC, allophycocyanin; BV, brilliant violet; FITC, fluorescein isothiocyanate; Cy, cyanine; PE, phycoerythrin; HRP, horseradish peroxidase;(DOCX) pone.0126034.s006.docx (17K) GUID:?2C669828-EC32-4D00-87AC-F43F7F3CB66A Abstract A variety of adjuvants fostering humoral immunity are known as of today. However, there is a lack of adjuvants or adjuvant strategies, which directly target T cellular effector functions and memory. We here decided whether systemically toxic cytokines such as IL-2 can be restricted to the.